D.D. and S.M.-D. acknowledge the BMBF Grant &#34;CellWiTaL: Reproducible cell systems for drug research - transfer layer-free laser printing of highly specific single cells in three-dimensional cellular structures&#34; Proposal Nr. 13N15874. D.D. and V.R.A. acknowledge the EU MSCA-COFUND Grant OSTASKILLS &#34;Holistic training of next-generation Osteoarthritis researches&#34; GA Nr. 101034412. All authors acknowledge Mrs. Beate Geyer for technical assistance.

NOTE: All the steps must be conducted using aseptic techniques.
1. Culture and pre-expansion of NHDFs
<ol>
	<li>Rapidly thaw the cryo-vial containing adult cryopreserved normal human dermal fibroblasts (NHDFs, 1 x 106 cells) at 37 &#176;C until they are almost defrosting.</li>
	<li>Slowly add 1 mL of prewarmed fibroblast growth medium 2 (ready-to-use kit including basal medium, 2% fetal calf serum (FCS), basic fibroblast growth factor (bFGF) and insulin) supplemen...

Three-Step Organoid Model for Tendon Research and Engineering

<ol>
	<li>Schneider, M., Angele, P., J&#228;rvinen, T. A. H., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+plan+for+Achilles:+Therapeutics+steering+the+fate+and+functions+of+stem+cells+in+tendon+wound+healing.">Rescue plan for Achilles: Therapeutics steering the fate and functions of stem cells in tendon wound healing.</a> Adv Drug Deliv Rev. 129, 352-375 (2018).</li><li>Steinmann, S., Pfeifer, C. G., Brochhausen, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+tendon+pathologies:+Triggers,+trails+and+end-state.">Spectrum of tendon pathologies: Triggers, trails and end-state.</a> Int J Mol Sci. 21 (3), 844 (2020).</li><li>Snedeker, J. G., Foolen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+injury+and+repair+-+A+perspective+on+the+basic+mechanisms+of+tendon+disease+and+future+clinical+therapy.">Tendon injury and repair - A perspective on the basic mechanisms of tendon disease and future clinical therapy.</a> Acta Biomater. 63, 18-36 (2017).</li><li>Lomas, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+in+scaffold-based+tendon+treatments.">The past, present and future in scaffold-based tendon treatments.</a> Adv Drug Deliv Rev. 84, 257-277 (2015).</li><li>Gomez-Florit, M., Labrador-Rached, C. J., Domingues, R. M. A., Gomes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tendon+microenvironment:+Engineered+in+vitro+models+to+study+cellular+crosstalk.">The tendon microenvironment: Engineered in vitro models to study cellular crosstalk.</a> Adv Drug Deliv Rev. 185, 114299 (2022).</li><li>Docheva, D., M&#252;ller, S. A., Majewski, M., Evans, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologics+for+tendon+repair.">Biologics for tendon repair.</a> Adv Drug Deliv Rev. 84, 222-239 (2015).</li><li>Kroner-Weigl, N., Chu, J., Rudert, M., Alt, V., Shukunami, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+Is+not+sufficient+to+facilitate+tenogenic+differentiation+of+dermal+fibroblasts+in+a+3D+organoid+model.">Dexamethasone Is not sufficient to facilitate tenogenic differentiation of dermal fibroblasts in a 3D organoid model.</a> Biomedicines. 11 (3), 772 (2023).</li><li>Chu, J., Pieles, O., Pfeifer, C. G., Alt, V., Morsczeck, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+follicle+cell+differentiation+towards+periodontal+ligament-like+tissue+in+a+self-assembly+three-dimensional+organoid+model.">Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.</a> Eur Cell Mater. 42, 20-33 (2021).</li><li>Yan, Z., Yin, H., Brochhausen, C., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aged+tendon+stem/progenitor+cells+are+less+competent+to+form+3D+tendon+organoids+due+to+cell+autonomous+and+matrix+production+deficits.">Aged tendon stem/progenitor cells are less competent to form 3D tendon organoids due to cell autonomous and matrix production deficits.</a> Front Bioeng Biotechnol. 8, 406 (2020).</li><li>Larkin, L. M., Calve, S., Kostrominova, T. Y., Arruda, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functional+evaluation+of+tendon-skeletal+muscle+constructs+engineered+in+vitro.">Structure and functional evaluation of tendon-skeletal muscle constructs engineered in vitro.</a> Tissue Eng. 12 (11), 3149-3158 (2006).</li><li>Schiele, N. R., Koppes, R. A., Chrisey, D. B., Corr, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cellular+fibers+for+musculoskeletal+soft+tissues+using+directed+self-assembly.">Engineering cellular fibers for musculoskeletal soft tissues using directed self-assembly.</a> Tissue Eng Part A. 19 (9-10), 1223-1232 (2013).</li><li>Florida, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structural+and+cellular+remodeling+of+engineered+bone-ligament-bone+constructs+used+for+anterior+cruciate+ligament+reconstruction+in+sheep.">In vivo structural and cellular remodeling of engineered bone-ligament-bone constructs used for anterior cruciate ligament reconstruction in sheep.</a> Connect Tissue Res. 57 (6), 526-538 (2016).</li><li>Mubyana, K., Corr, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+uniaxial+tensile+strain+enhances+the+mechanical+properties+of+engineered,+scaffold-free+tendon+fibers.">Cyclic uniaxial tensile strain enhances the mechanical properties of engineered, scaffold-free tendon fibers.</a> Tissue Eng Part A. 24 (23-24), 1808-1817 (2018).</li><li>Brassard, J. A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+stem+cell+self-organization+to+build+better+organoids.">Engineering stem cell self-organization to build better organoids.</a> Cell Stem Cell. 24 (6), 860-876 (2019).</li><li>Kim, J., Koo, B. K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+model+systems+for+human+biology+and+medicine.">Human organoids: model systems for human biology and medicine.</a> Nat Rev Mol Cell Biol. 21 (10), 571-584 (2020).</li><li>Hsieh, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+comparison+of+2D-cell+culture+and+3D-cell+sheets+of+scleraxis-programmed+bone+marrow+derived+mesenchymal+stem+cells+to+primary+tendon+stem/progenitor+cells+for+tendon+repair.">In vitro comparison of 2D-cell culture and 3D-cell sheets of scleraxis-programmed bone marrow derived mesenchymal stem cells to primary tendon stem/progenitor cells for tendon repair.</a> Int J Mol Sci. 19 (8), 2272 (2018).</li><li>Chu, J., Lu, M., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+tendons:+A+concise+review+on+the+potential+of+dermal+fibroblasts.">Rebuilding tendons: A concise review on the potential of dermal fibroblasts.</a> Cells. 9 (9), 2047 (2020).</li><li>Gaspar, D., Spanoudes, K., Holladay, C., Pandit, A., Zeugolis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+cell-based+therapies+for+tendon+repair.">Progress in cell-based therapies for tendon repair.</a> Adv Drug Deliv Rev. 84, 240-256 (2015).</li><li>Sacco, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+dermal+fibroblasts+as+major+determinant+of+variability+in+cell+reprogramming.">Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.</a> J Cell Mol Med. 23 (6), 4256-4268 (2019).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+predominant+tenogenic+phenotype+in+human+dermal+fibroblasts+via+synergistic+effect+of+TGF-&#946;+and+elongated+cell+shape.">Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-&#946; and elongated cell shape.</a> Am J Physiol Cell Physiol. 310 (5), C357-C372 (2016).</li><li>Pryce, B. A., Watson, S. S., Murchison, N. D., Staverosky, J. A., D&#252;nker, N., Schweitzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+and+maintenance+of+tendon+progenitors+by+TGFbeta+signaling+are+essential+for+tendon+formation.">Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation.</a> Development. 136 (8), 1351-1361 (2009).</li><li>Pattappa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physioxia+has+a+beneficial+effect+on+cartilage+matrix+production+in+interleukin-1+beta-inhibited+mesenchymal+stem+cell+chondrogenesis.">Physioxia has a beneficial effect on cartilage matrix production in interleukin-1 beta-inhibited mesenchymal stem cell chondrogenesis.</a> Cells. 8 (8), 936 (2019).</li><li>Shafiee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+physiologically+relevant+skin+organoids+from+human+induced+pluripotent+stem+cells.">Development of physiologically relevant skin organoids from human induced pluripotent stem cells.</a> Small. 2304879, (2023).</li><li>Zeiger, A. S., Hinton, B., Van Vliet, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+the+dish+makes+a+difference:+quantitative+comparison+of+polystyrene+culture+surfaces.">Why the dish makes a difference: quantitative comparison of polystyrene culture surfaces.</a> Acta Biomater. 9 (7), 7354-7361 (2013).</li><li>Koh, T. M., Feih, S., Mouritz, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengthening+mechanics+of+thin+and+thick+composite+T-joints+reinforced+with+z-pins.">Strengthening mechanics of thin and thick composite T-joints reinforced with z-pins.</a> Compos Part A Appl Sci. 43 (8), 1308-1317 (2012).</li><li>Gelberman, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+administration+of+ASCs+and+BMP-12+promotes+an+M2+macrophage+phenotype+and+enhances+tendon+healing.">Combined administration of ASCs and BMP-12 promotes an M2 macrophage phenotype and enhances tendon healing.</a> Clin Orthop Relat Res. 475 (9), 2318-2331 (2017).</li><li>Hofer, M., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+organoids.">Engineering organoids.</a> Nat Rev Mater. 6 (5), 402-420 (2021).</li><li>Driehuis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+organoids+recapitulate+disease+and+allow+personalized+drug+screening.">Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.</a> Proc Natl Acad Sci U S A. 116 (52), 26580-26590 (2019).</li><li>Yan, H. H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+human+gastric+cancer+organoid+biobank+captures+tumor+subtype+heterogeneity+and+enables+therapeutic+screening.">A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.</a> Cell Stem Cell. 23 (6), 882-897 (2018).</li></ol>

The results demonstrated in this study provide valuable insights into the establishment and characterization of the NHDF 3D organoid model for studying T/L tissues. The 3-step protocol led to the formation of 3D rod-like organoids that exhibit typical features of T/L niche. This model was previously reported in Kroner-Weigl et al. 20237 and demonstrated in great detail here.
The phase-contrast images presented in Figure 2 showed th...

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure and function. Over the past few years, researchers have strived to better understand the intricate nature of T/L in order to seek effective treatments for T/L disorders3,4,5. T/L are distinguished by a hierarchically organized, extracellular matrix (ECM)-dominated structure, composed primarily of type I collagen fibers and proteoglycans, a feature that is difficult to be replicated in vitro6. Traditional two-dimensional (2D) cell culture models fail to capture the characteristic three-dimensional (3D) organization of T/L tissues, limiting their translational potential as well as hindering the innovative progress in the field of T/L regeneration.
Recently, the development of 3D organoid models has offered new possibilities for advancing basic research and scaffolds-free tissue engineering of various tissue types7,8,9,10,11,12,13. For example, to investigate myotendinous junction, Larkin et al. 2006 developed 3D skeletal muscle constructs together with self-organized tendon segments derived from rat tail tendon10. Moreover, Schiele et al. 2013, by using micromachined fibronectin-coated growth channels, directed the self-assembly of human dermal fibroblasts to form cellular fibers without the assistance of 3D scaffold, an approach that can capture key traits of embryonic tendon development11. In the study by Florida et al. 2016, bone marrow stromal cells were first expanded into bone and ligament lineages, next used to generate self-assembled monolayer cell sheets, which were then implemented to create a multiphasic bone-ligament-bone construct mimicking the native anterior cruciate ligament, a model aiming at improved understanding of ligament regeneration12. To elucidate tendon mechanotransduction processes, Mubyana et al. 2018 utilized a scaffold-free methodology by which single tendon fibers were created and subjected to mechanical loading protocol13. Organoids are self-organized 3D structures that mimic the native architecture, microenvironment, and functionality of tissues. 3D organoid cultures provide a more physiologically relevant model for studying tissue and organ biology as well as pathophysiology. Such models can also be used to induce tissue-specific differentiation of different stem/progenitor cell types14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach9,16. Alternative cell sources can be implemented for the organoid assembly and stimulated toward tenogenic differentiation. One relevant cell type used for demonstration in this study is dermal fibroblasts7,17,18. These cells are easily accessible through a skin biopsy procedure, which is less invasive compared to bone marrow puncture or liposuction and can be multiplied fairly quickly to large numbers due to their good proliferative capacity. In contrast, more specialized cell types, such as T/L-resident fibroblasts, are more challenging to isolate and expand. Therefore, dermal fibroblasts were also used as a starting point for cell reprogramming technologies towards induced pluripotent embryonic stem cells19. Subjecting dermal fibroblasts to specific 3D culture conditions and signaling cues, such as transforming growth factor-beta 3 (TGF&#223;3), which has been reported to act as a key regulator of various cellular processes, including the formation and maintenance of T/L, can potentiate their in vitro tenogenic differentiation leading to the expression of tendon-specific genes and the deposition of T/L-typical ECM20,21.
Here, we describe and demonstrate a previously established and implemented 3-step (2D expansion, 2D stimulation, and 3D maturation) organoid protocol using commercially available normal adult human dermal fibroblasts (NHDFs) as a cell source, offering a valuable model for studying in vitro tenogenesis7. Despite the fact that this model is not equivalent to in vivo T/L tissue, it still provides a more physiologically relevant system that can be used for investigating cellular differentiation mechanisms, mimicking T/L pathophysiology in vitro, and establishing T/L personalized medicine and drug screening platforms. Moreover, in the future, studies can evaluate whether the 3D organoids are suitable for scaffold-free T/L engineering by further optimization as well as utilizable for the development of scaled-up mechanically robust constructs that closely resemble the dimensions and structural and biophysical properties of native T/L tissues.

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			<td>Ascorbic acid&#160;&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;&#160;</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm adherent cell culture dish</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430167</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm non-adherent petri dish&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430591</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-medium</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands&#160;&#160;</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold standard&#160;</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands</td>
			<td>4557</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Sucrose&#160;</td>
			<td>AppliChem Avantor VWR International GmbH, Darmstadt, Germany</td>
			<td>A2211</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose medium&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>DMEM-HA</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>DMEM-LPXA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>&#160;Anprotec, Bruckberg, Germany&#160;</td>
			<td>AC-SM-0027</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth medium 2&#160;</td>
			<td>PromoCell, Heidelberg, Germany&#160;&#160;</td>
			<td>C-23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope with high resolution camera</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Zeiss Axio Observer with&#160; Axiocam 506</td>
		</tr>
		<tr>
			<td>MEM amino acids&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>NEAA-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal pins&#160;</td>
			<td>EntoSphinx, Pardubice, Czech Republic&#160;</td>
			<td>04.31</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal human dermal fibroblasts&#160;</td>
			<td>&#160;PromoCell, Heidelberg, Germany&#160;</td>
			<td>C-12302</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>&#160;AppliChem, Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>A3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Gibco, Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#223;3&#160;</td>
			<td>R&#38;D Systems, Wiesbaden, Germany&#160;</td>
			<td>&#160;8420-B3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0,05% DPBS&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>&#160;TRY-1B</td>
			<td></td>
		</tr>
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</table>

demonstration of self-assembled cell sheet culture and manual generation of a 3d tendon/ligament-like organoid by using human dermal fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Author Spotlight: Advancing Tendon Tissue Engineering with 3D Organoid Models

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

We demonstrate a previous established and implement three-step organoid protocol using commercially available, normal human dermal fibroblasts as a cell source. The 3D organoid model holds several advantage for tissue engineering and regenerative medicine application. It mimics closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of tendon tissues.The established 3D tendon-ligament organoid approach can be used as a model for tendon basic and applied research in the following areas. In vitro tenogenesis, disease mechanism, drug testing, and scaffold-free tendon tissue engineering.

The 3D T/L organoid model was previously established and demonstrated here by implementing commercially purchased NHDF (n=3, 3 organoids per donor, NHDF were used at passages 5-8). The model workflow is summarized in Figure 1. Figure 2 shows representative phase-contrast images of NHDF culture during the pre-expansion in T-75 flasks (Figure 2A) as well as at the beginning and after 5 days of culture in the 2D expansion step in 10 cm...

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

demonstration-self-assembled-cell-sheet-culture-manual-generation-3d

Research

JoVE Journal

Bioengineering

667 vistas.  University of Würzburg. Demostramos un protocolo de organoides de tres pasos previamente establecido e implementado utilizando fibroblastos dérmicos humanos normales disponibles comercialmente como fuente de células. El modelo de organoide 3D tiene varias ventajas para la ingeniería de tejidos y la aplicación de la medicina regenerativa. Imita de cerca la composición y organización celular, así como las interacciones célula a célula y célula a matriz de los tejidos tendinosos.El enfoque establecido ...